Targeted Protein Detection Using an All-in-One Mass Spectrometry

Jul 31, 2017 - We developed a simple 3D printed cartridge for mass spectrometry (MS) targeted detection of plasma proteins, including post-translation...
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Targeted Protein Detection Using an All-in-One Mass Spectrometry Cartridge Chengsen Zhang,† Trevor Glaros,‡ and Nicholas E. Manicke*,† †

Department of Chemistry and Chemical Biology, Indiana UniversityPurdue University Indianapolis, Indianapolis, Indiana 46202, United States ‡ BioSciences Division, BioDefense Branch, US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland 21010, United States S Supporting Information *

detection of proteins from complex biofluids such as plasma. Selective enrichment of protein targets is performed oncartridge passively by capillary action and gravity with no pumping. Detection of the intact protein targets then proceeds via ionization of the protein target using a built-in spray substrate consisting of carbon-nanotube (CNT) coated porous polyethylene. The distinguishing feature of this approach is its simplicity and low cost, in effect a combination of inexpensive lateral flow assays with mass spectrometry. Three applications of this cartridge are described for clinically significant problems: (1) identification of T45S variant of apolipoprotein c1, a polymorphism associated with body mass index and obesity; (2) relative quantitation of hemoglobin (Hb) A1C, a marker of diabetes; and (3) identification of sequence variants of transthyretin, which are markers of transthyretin-related hereditary amyloidosis. A 3D printed cartridge (Figure 1) was developed to perform the entire analyte preconcentration and ionization process. An absorbent waste pad in the base of the cartridge draws the sample through the antibody column, which consisted of a

ABSTRACT: We developed a simple 3D printed cartridge for mass spectrometry (MS) targeted detection of plasma proteins, including post-translational modifications (PTMs). The cartridge uses an integrated antibody enrichment column to preconcentrate the protein target as well as a novel built-in substrate to ionize the protein targets for MS detection. We show several examples of using this cartridge to perform rapid detection of clinically significant proteoforms from plasma samples.

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rotein molecules are measured in clinical diagnostic laboratories as tumor markers, acute or chronic disease markers, risk or prognostic markers, and hormones. Immunoassays have been successfully used for decades to detect protein analytes in clinical laboratories, but there remain limitations of this approach. Monoclonal antibodies are expensive to develop and manufacture. Moreover, there are well recognized analytical limitations; for example, most immunoassays do not distinguish between the various proteoforms and variants of a protein present in clinical samples.1 Targeted enrichment of proteins followed by MS detection is a widely used approach to detect different proteoforms. Enrichment is generally performed using antibodies because of their ability to selectively enrich target proteins from proteinrich matrices such as plasma. Mass spectrometric immunoassay (MSIA) uses antibody coated beads packed into a pipet tip2 followed by detection of intact proteins by MALDI-MS. Another approach is antibody modification of the MALDI target.3−5 In SISCAPA (stable isotope standards and capture by antipeptide antibodies) and immuno-MALDI, enrichment is performed at the peptide level after digestion.6,7 Peptide level enrichment gives better quantitation and improved sensitivity. However, limited sequence coverage complicates detection of PTMs and sequence variants. Development of a simple MS assay capable of resolving proteoforms would have a significant impact on protein detection, including clinical testing. We previously reported simple, single-use MS cartridges for the analysis of drugs which integrated sample preparation, sample storage as a dried spot, and analyte ionization via paper spray, in which paper is used as an inexpensive solid support for extraction, fluid transport, and electrospray ionization.8,9 Here, we describe the development of a mass spectrometry cartridge designed for targeted © 2017 American Chemical Society

Figure 1. Schematic diagram and photograph of the antibody cartridge. The inserted photograph shows the assembled cartridge in the sample loading/wash. The antibody column extracted target protein from the plasma, and a CNT-coated porous polyethylene spray tip was used for substrate supported electrospray. Received: May 30, 2017 Published: July 31, 2017 10996

DOI: 10.1021/jacs.7b05571 J. Am. Chem. Soc. 2017, 139, 10996−10999

Communication

Journal of the American Chemical Society plastic tube packed with glass fiber membranes coated with latex bead−antibody conjugates. All parts were assembled together, and the column holder was slid along the groove between lid and base to switch the position of the antibody column from above the waste pad, where the sample addition and washing steps occurred, to above the spray substrate where ionization by substrate supported electrospray ionization occurred. MS detection was performed on a quadrupoleorbitrap mass spectrometer in full scan mode. A number of reports describe the use of paper spray MS for the detection of drugs and other small molecules from blood.10,11 The detection efficiency of proteins by paper spray is relatively poor, however. A paper spray based immunoassay was reported in which a charged probe released from the bound antibody was detected by MS.12 Although this approach has better sensitivity because a small molecule is used for detection, the selectivity is ultimately limited by the antibody. Detection of the probe cannot give any information on the antigen such as PTMs. Improved protein ionization has been reported using paper coated with CNTs13 as well as size exclusion membranes.14 Carbon nanotube coated paper has also been utilized for low voltage paper spray.15 We investigated CNT treated paper and as well as a novel substrate, CNT treated porous polyethylene (PE). Photographs and scanning electron microscope images of the spray substrates are shown in Figure S1. Detection limits of three proteins were improved by coating or dispersing chromatography paper with singlewalled CNTs (Table S1). The CNT-coated PE even further improved protein detection; CNT-coated PE detection limits were between 10 and 100 times lower compared to the best obtained using CNT treated paper. Detection limits of the protein standards were improved by a factor of 50−1000 compared to typical paper spray. Mass spectra obtained using paper and CNT-coated PE for a standard protein solution are shown in Figure 2.

Figure 3. Taylor cone generated from CNT-PE and paper. The diameter of the Taylor cone generated from the sharp point of CNTPE was much smaller compared with paper. CNT-coated paper is pictured in Figure S3.

To achieve selective and sensitive detection of target proteins from biofluids, we utilized an antibody column integrated into the cartridge. In this method, protein preconcentration was performed using a membrane containing latex bead−antibody conjugates. The plasma sample was added to the antibody column where it flowed through by a combination of capillary action and gravity. Excess sample was absorbed into a built-in waste pad in the bottom cartridge, while the target protein was retained on the latex bead conjugates. A washing step was performed by subsequently adding water to the preconcentration column, where it wicked into the waste pad. The protein analyte was then detected by sliding the antibody column to the elution position above the spray substrate and adding 1:1 methanol/water with 2% acetic acid, which acted as both the extraction solution and the spray solvent. Over 90% of captured protein was eluted in the case of cytochrome c (Supporting Information). We used polyclonal antibodies as opposed to monoclonal antibodies. While monoclonal antibodies are generally preferred in immunoassays because of their higher selectivity, polyclonal antibodies have a number of advantages for this application. Polyclonal antibodies have higher affinity compared to monoclonal antibodies, they bind to multiple different epitopes which minimizes the risk that a particular proteoform or variant will not bind, and they are cheaper than monoclonal antibodies. The lower selectivity of polyclonal antibodies is not a factor in this application because of the use of MS detection. This approach does necessitate that antibodies against the target protein be available. Antibodies are also the most expensive component of the cartridge. However, use of antibodies in this cartridge are still significantly cheaper with faster development times than traditional immunoassays. Because MS detection is used, high selectivity monoclonal antibodies are not required, which decreases the cost and time associated with antibody generation. We investigated three clinically significant applications of the MS protein detection cartridge. Apolipoprotein C1 (ApoC1) T45S variant is a naturally occurring amino acid polymorphism that has been associated with elevated body mass index and diabetes.17 As shown in Figure 4a, three ApoC1 species were identified in the MS spectrum obtained from a pooled human plasma sample, including full-length ApoC1 (6.6 kDa), truncated ApoC1(minus amino-terminal Thr-Pro, 6.4 kDa), and truncated T45S variant (Δm = −14.03 Da). Detection of the T45S variant in the pooled plasma sample indicated that some of the plasma came from individuals with the ApoC1 S45 allele (Figure 4b). The protein cartridge was also used to analyze plasma from a single donor; the mass spectrum indicates that the individual donor did not have this allele

Figure 2. Full MS spectra obtained by ionizing 10 μg/mL cytochrome c using paper spray (a) and the CNT-coated PE substrate (b). Spectra for two other proteins are in Figure S2.

The Taylor cone generated from the CNT-coated PE tip was significantly smaller compared to paper (Figure 3). The size of the Taylor cone correlates with droplet size, and smaller electrospray droplets correlate with better ionization efficiency.16 The improved protein detection from the CNTcoated PE substrate therefore may arise from balancing the surface energy of the substrate with that of the solvent to generate smaller droplets and more efficient ionization. The substrate must be sufficiently wettable to allow wicking by a polar solvent, necessary to drive fluid and analyte transport, but not so wettable that the Taylor cone is large. The CNT−PE substrate was also found to retain less protein than paper, but the difference was small compared to the improvement in detection limits (Supporting Information). 10997

DOI: 10.1021/jacs.7b05571 J. Am. Chem. Soc. 2017, 139, 10996−10999

Communication

Journal of the American Chemical Society

Figure 4. MS protein detection cartridge analysis of human plasma apolipoprotein c1 (ApoC1). (a) Full MS spectrum obtained from the pooled human plasma sample showing the ApoC1 full-length, truncated, and truncated T45S variant. Narrower MS scan range shows the truncated ApoC1 + 6 ions obtained from (b) pooled human plasma and (c) individual donated plasma.

Figure 5. Antibody cartridge analysis of human plasma hemoglobin (Hb). (a) Full MS spectrum shows the Hb α-/β-chain ions with charge state from +11 to +20. SIM spectra (m/z scan window 1.2 Da) show the relative intensities of nonglycated (b) and glycated (c) Hb.

step, succeeded in eliminating the S-cysteine and S-glutathione modifications (Figure S4), although the S-sulfonation remained. Four different TTR mutants in six ATTR clinical samples were clearly identified and differentiated from wt TTR. Figure 6 shows the charge-state deconvoluted mass spectra obtained from a wt sample and a representative ATTR sample. The spectrum from the ATTR sample indicates the patient is heterozygous for wild type TTR and a TTR variant with a Δm of 32.00 Da. This mass shift is consistent with substitution of

(Figure 4c). In comparison with calculated accurate masses, mass error for detected T45S variant was 0.0056 Da. These results indicate the method has good potential for rapid detection of the ApoC1 T45S variant. Glycated hemoglobin is a stable minor Hb variant formed in vivo by the nonenzymatic, covalent attachment of glucose. Relative quantitation of a particular Hb fraction, HbA1c, has been used as a marker of glycemia control in diabetes for decades.18 As shown in Figure 5, several hemoglobin species were identified using the protein detection cartridge, such as nonglycated α-/β-chains (Figure 5a) and glycated α-/β-chains (Figure 5a insert spectrum). The Hb and glycated Hb were monitored using single ion monitoring (SIM) of the +15 charge state as shown in Figure 5b and 5c. The mass error for the detected nonglycated Hb β-chain and glycated Hb β-chain were 0.012 Da and −0.079 Da, respectively. The relative intensity of glycated Hb was ∼7% compared to unglycated Hb, which is similar to the expected proportion.19,20 The results indicate the method has potential for relative quantitation of Hb and HbA1c. Transthyretin (TTR) is a 55 kDa homotetramer protein that transports thyroxine and retinol in blood and cerebrospinal fluid. Misfolding and aggregation of wild-type (wt) and mutant transthyretin protein (TTR) is the cause of transthyretin amyloidosis (ATTR), a severe and fatal disease characterized by abnormal deposits of TTR amyloids. Mass spectrometry is used to screen for ATTR.20 The antibody cartridge was successfully used to detect wt TTR as well as various TTR mutants from human plasma samples. Figure S4 shows the mass spectrum obtained for the +10 charge state of wt TTR. The unmodified TTR was a relatively minor component; the cysteine, glutathione, and sulfonate PTMs were all more intense. To reduce the complexity of the mass spectrum, we spiked the reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) into the spray solvent. This procedure, performed on-cartridge in the extraction/spray solvent without a separate

Figure 6. Charge-state deconvoluted MS spectra obtained by antibody cartridge analysis of human plasma from a wt sample (a) and a ATTR plasma sample (b). Only wild type TTR, including free thiol wt-TTR and S-sulfonation wt-TTR, can be detected from normal sample, while a mutant TTR with Δm = 32.0 Da was identified from the ATTR sample. 10998

DOI: 10.1021/jacs.7b05571 J. Am. Chem. Soc. 2017, 139, 10996−10999

Communication

Journal of the American Chemical Society

(17) Kasthuri, R. S.; McMillan, K. R.; Flood-Urdangarin, C.; Harvey, S. B.; Wilson-Grady, J. T.; Nelsestuen, G. L. Int. J. Obes. 2007, 31, 1334. (18) Gillery, P. Clin. Chem. Lab. Med. 2013, 51, 65. (19) Weykamp, C. Ann. Lab. Med. 2013, 33, 393. (20) Lim, A.; Prokaeva, T.; McComb, M. E.; O’Connor, P. B.; Théberge, R.; Connors, L. H.; Skinner, M.; Costello, C. E. Anal. Chem. 2002, 74, 741. (21) Saraiva, M. J. M. J. Peripher. Nerv. Syst. 1996, 1, 179.

valine-30 for methionine, the most common mutation found in TTR associated neuropathy.21 In summary, we describe a simple, all-in-one 3D printed cartridge which enables targeted protein detection from plasma samples by mass spectrometry. The unique feature of this device is that both protein enrichment and ionization occur from one device. Because of the simplicity of the analytical procedure, this cartridge better enables MS-based detection of protein targets, which is important in clinical diagnostics and other fields. While MS is expensive relative to traditional immunoassay detection methods, mass spectrometry is better suited for distinguishing post-translational modifications, sequence variants, and other proteoforms.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b05571. Materials, full experimental details, supplemental Figures S1−S4 and Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Chengsen Zhang: 0000-0001-5674-0794 Trevor Glaros: 0000-0001-9922-6330 Nicholas E. Manicke: 0000-0002-2296-0497 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Thermo Scientific for funding and other support. We also thank Merrill Benson MD, IU School of Medicine, for supplying the ATTR samples.

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REFERENCES

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DOI: 10.1021/jacs.7b05571 J. Am. Chem. Soc. 2017, 139, 10996−10999